Mycobacterium tuberculosis (Mtb), the causative agent of the pulmonary infection tuberculosis, has evaded eradication from the human population due to its extraordinary ability to both persist in latently infected individuals and to generate antibiotic-tolerant persister cells. We hypothesize that different molecular mechanisms account for these two types of Mtb persistence and propose novel high-throughput genetic screens to identify and characterize these mechanisms. We will use a new technology, Tn-seq, that simultaneously identifies and quantifies transposon (Tn) insertion mutants within large random Tn mutant pools by massively parallel sequencing of Tn-genome junctions. Tn-seq has previously been used to analyze fitness of bacterial Tn mutants in various culture conditions in vitro and in some animal infection models in vivo. But Tn-seq has limited utility for studying growth conditions or infection models in which there are narrow colonization bottlenecks. We propose to adapt the Tn-seq method using bar-code sequence tags and multi-plexing to enable cost-effective analysis of smaller Tn mutant pools. We will use Tn-seq combined with infection of genetically modified mice to define on a genome- wide scale the factors that Mtb requires for persistence in the face of specific host adaptive immune defenses. We will additionally use an in vitro antibiotic selection strategy combined with Tn-seq to define factors that Mtb requires for optimal formation of antibiotic-tolerant persister cells. Ultimately, we will expand these results o relevant animal infection models, to demonstrate that the Mtb persistence factors we identify are viable drug targets. Therapeutics targeting these persistence factors would represent novel approaches to tuberculosis control that would sensitize Mtb either to natural host immune defenses or to existing antibiotics.